NF-κb inhibitors

ABSTRACT

The present invention provides novel compounds and methods for using them to treat diseases with aminothiophene inhibitors of IKK-β phosphorylation of IκB. In so doing these aminothiophene inhibitors block pathological activation of transcription factor NF-κB in which diseases excessive activation of NF-κB is implicated.

This application is a 371 of International Application No.PCT/US03/16876, filed May 29, 2003, which claims the benefit of U.S.Provisional Application No. 60/386,557, filed Jun. 6, 2002.

FIELD OF THE INVENTION

This invention relates in general to a method of inhibiting pathologicalactivation of the transcription factor NF-κB (nuclear factor-κB) usingamino-benzothiophene compounds. Such methods are particularly useful fortreating diseases in which activation of NF-κB is implicated. Morespecifically, these methods may be used for inhibiting IKK-β (IκBkinase-β, also known as IKK-2) phosphorylation of IκB (inhibitoryprotein κB)—which prevents subsequent degradation and activation ofNF-κB dimers. Such methods are useful in the treatment of a variety ofdiseases associated with NF-κB activation including inflammatory andtissue repair disorders; particularly rheumatoid arthritis, inflammatorybowel disease, asthma and COPD (chronic obstructive pulmonary disease)osteoarthritis; osteoporosis and fibrotic diseases; dernatosis,including psoriasis, atopic dermatitis and ultraviolet radiation(UV)-induced skin damage; autoimmune diseases including systemic lupuseythematosus, multiple sclerosis, psoriatic arthritis, alkylosingspondylitis, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer,including Hodgkins disease, cachexia, inflammation associated withinfection and certain viral infections, including acquired immunedeficiency syndrome (AIDS), adult respiratory distress syndrome, AtaxiaTelangiestasia.

BACKGROUND OF THE INVENTION

Recent advances in scientific understanding of the mediators involved inacute and chronic inflammatory diseases and cancer have led to newstrategies in the search for effective therapeutics. Traditionalapproaches include direct target intervention such as the use ofspecific antibodies, receptor antagonists, or enzyme inhibitors. Recentbreakthroughs in the elucidation of regulatory mechanisms involved inthe transcription and translation of a variety of mediators have led toincreased interest in therapeutic approaches directed at the level ofgene transcription.

Nuclear factor κB (NF-κB) belongs to a family of closely related dimerictranscription factor complexes composed of various combinations of theRel/NF-κB family of polypeptides. The family consists of five individualgene products in mammals, RelA (p65), NF-κB1 (p50/p105), NF-κB2(p49/p100), c-Rel, and RelB, all of which can form hetero- orhomodimers. These proteins share a highly homologous 300 amino acid “Relhomology domain” which contains the DNA binding and dimerizationdomains. At the extreme C-terminus of the Rel homology domain is anuclear translocation sequence important in the transport of NF-κB fromthe cytoplasm to the nucleus. In addition, p65 and cRel possess potenttransactivation domains at their C-terminal ends.

The activity of NF-κB is regulated by its interaction with a member ofthe inhibitor IκB family of proteins. This interaction effectivelyblocks the nuclear localization sequence on the NF-κB proteins, thuspreventing migration of the dimer to the nucleus. A wide variety ofstimuli activate NF-κB through what are likely to be multiple signaltransduction pathways. Included are bacterial products (LPS), someviruses (HIV-1, HTLV-1), inflammatory cytokines (TNFα, IL-1),environmental and oxidative stress and DNA damaging agents. Apparentlycommon to all stimuli however, is the phosphorylation and subsequentdegradation of IκB. IκB is phosphorylated on two N-terminal serines bythe recently identified IκB kinases (IKK-α and IKK-β). Site-directedmutagenesis studies indicate that these phosphorylations are criticalfor the subsequent activation of NF-κB in that once phosphorylated theprotein is flagged for degradation via the ubiquitin-proteasome pathway.Free from IκB, the active NF-κB complexes are able to translocate to thenucleus where they bind in a selective manner to preferred gene-specificenhancer sequences. Included in the genes regulated by NF-κB are anumber of cytokines and chemokines, cell adhesion molecules, acute phaseproteins, immunoregualtory proteins, eicosanoid metabolizing enzymes andanti-apoptotic genes.

It is well-known that NF-κB plays a key role in the regulated expressionof a large number of pro-inflammatory mediators including cytokines suchas TNF, IL-1β, IL-6 and IL-8, cell adhesion molecules, such as ICAM andVCAM, and inducible nitric oxide synthase (iNOS). Such mediators areknown to play a role in the recruitment of leukocytes at sites ofinflammation and in the case of iNOS, may lead to organ destruction insome inflammatory and autoimmune diseases.

The importance of NF-κB in inflammatory disorders is furtherstrengthened by studies of airway inflammation including asthma, inwhich NF-κB has been shown to be activated. This activation may underliethe increased cytokine production and leukocyte infiltrationcharacteristic of these disorders. In addition, inhaled steroids areknown to reduce airway hyperresponsiveness and suppress the inflammatoryresponse in asthmatic airways. In light of the recent findings withregard to glucocorticoid inhibition of NF-κB, one may speculate thatthese effects are mediated through an inhibition of NF-κB.

Further evidence for a role of NF-κB in inflammatory disorders comesfrom studies of rheumatoid synovium. Although NF-κB is normally presentas an inactive cytoplasmic complex, recent immunohistochemical studieshave indicated that NF-κB is present in the nuclei, and hence active, inthe cells comprising rheumatoid synovium. Furthermore, NF-κB has beenshown to be activated in human synovial cells in response to stimulationwith TNF-α or IL-1β. Such a distribution may be the underlying mechanismfor the increased cytokine and eicosanoid production characteristic ofthis tissue. See Roshak, A. K., et al., J. Biol. Chem., 271, 31496-31501(1996). Expression of IKK-β has been shown in synoviocytes of rheumatoidarthritis patients and gene transfer studies have demonstrated thecentral role of IKK-β in stimulated inflammatory mediator production inthese cells. See Aupperele et al. J. Immunology 1999. 163:427-433 andAupperle et al. J. Immunology 2001; 166:2705-11. More recently, theintra-articular administration of a wild type IKK-β adenoviral constructwas shown to cause paw swelling while intra-articular administration ofdominant-negative IKK-β inhibited adjuvant-induced arthritis in rat. SeeTak et al. Arthritis and Rheumatism 2001; 44:1897-1907.

The NF-κB/Rel and IκB proteins are also likely to play a key role inneoplastic transformation and metastasis. Family members are associatedwith cell transformation in vitro and in vivo as a result ofoverexpression, gene amplification, gene rearrangements ortranslocations. In addition, rearrangement and/or amplification of thegenes encoding these proteins are seen in 20-25% of certain humanlymphoid tumors. Further, NF-κB is activated by oncogenic ras, the mostcommon defect in human tumors and blockade of NF-κB activation inhibitsras mediated cell transformation. In addition, a role for NF-κB in theregulation of apoptosis has been reported, strengthening the role ofthis transcription factor in the regulation of tumor cell proliferation.TNF, ionizing radiation and DNA damaging agents have all been shown toactivate NF-κB which in turn leads to the upregulated expression ofseveral anti-apoptotic proteins. Conversely, inhibition of NF-κB hasbeen shown to enhance apoptotic-killing by these agents in several tumorcell types. As this likely represents a major mechanism of tumor cellresistance to chemotherapy, inhibitors of NF-κB activation may be usefulchemotherapeutic agents as either single agents or adjunct therapy.Recent reports have implicated NF-κB as an inhibitor of skeletal celldifferentiation as well as a regulator of cytoline-induced musclewasting (Guttridge et al. Science; 2000; 289: 2363-2365.) furthersupporting the potential of NF-κB inhibitors as novel cancer therapies.

Several NF-κB inhibitors are described in C. Wahl, et al. J. Clin.Invest. 101(5), 1163-1174 (1998), R. W. Sullivan, et al. J. Med. Chem.41, 413-419 (1998), J. W. Pierce, et al. J. Biol. Chem. 272, 21096-21103(1997)

The marine natural product hymenialdisine is known to inhibit NF-κB.Roshak, A., et al., JPET, 283, 955-961 (1997). Breton, J. J andChabot-Fletcher, M. C., JPET, 282, 459-466 (1997).

Additionally, patent applications have been filed on aminothiopheneinhibitors of the IKK-2, see Callahan, et al., WO 2002030353; Baxter, etal., WO 2001058890, Faull, et al., WO 2003010158; Griffiths, et al.,WO2003010163; Fancelli, et al., WO 200198290; imidazole inhibitors ofIKK-2, see Callahan, et al., WO 200230423; anilinophenylpyrimidineinhibitors of IKK-2, see Kois, et al., WO 2002046171; β-carbolineinhbitors of IKK-2, see Ritzeler, et al., WO 2001068648, Ritzeler, etal., EP 1134221; Nielsch, et al. DE 19807993; Ritzeler, et al., EP1209158; indole inhbitors of IKK-2, see Ritzeler, et al., WO 2001030774;benzimidazole inhibitors of the IKK-2, see Ritzeler, et al., DE19928424; Ritzeler et al, WO 2001000610; aminopyridine inhibitors ofIKK-2, see Lowinger, et al, WO2002024679; Murata, et al, WO 2002024693;Murata, et al., WO2002044153;pyrazolaquinazoline inhibitors of IKK-2,see Beaulieu, et al., WO2002028860; Burke et al, WO2002060386, Burke, etal. U.S. 20030022898; quinoline inhibitors of IKK-2, Browner, et al.,WO2002041843, Browner, et al., U.S. 20020161004 andpyridylcyanoguanidine inhibitors of IKK-2, see Bjorkling, et al., WO2002094813, Binderup et al, WO 2002094322 and Madsen, et al., WO200294265. The natural products staurosporine, quercetin, K252a andK252b have been shown to be IKK-2 inhibitors, see Peet, G. W. and Li, J.J. Biol. Chem., 274, 32655-32661 (1999) and Wisniewski, D., et al.,Analytical Biochem. 274, 220-228 (1999). Synthetic inhibitors of IKK-2have also been described, see Burke, et al. J. Biol. Chem., 278,1450-1456 (2003) and Murata, et al., Bioorg. Med. Chem. Lett., 13,913-198 (2003) have described IKK-2 inhibitors.

U.S. Pat. No. 3,963,750 describes the preparation of certainaminothiophenes.

SUMMARY OF THE INVENTION

The present invention involves novel compounds and novel methods ofinhibiting the activation transcription factor NF-κB using the presentcompounds.

An object of the present invention is to provide a method for treatingdiseases which may be therapeutically modified by altering the activityof transcription factor NF-κB.

Accordingly, in the first aspect, this invention provides apharmaceutical composition comprising a compound according to Formula I.

In another aspect, this invention provides a method of treating diseasesin which the disease pathology may be therapeutically modified byinhibiting phosphorylation and subsequent degradation of IκB by IKK-β.

In still another aspect, this invention provides a method of treatingdiseases in which the disease pathology may be therapeutically modifiedby inhibiting pathological activation of NF-κB.

In a particular aspect, this invention provides methods for treating avariety of diseases associated with NF-κB activation includinginflammatory and tissue repair disorders, particularly rheumatoidarthritis, inflammatory bowel disease, asthma and COPD (chronicobstructive pulmonary disease) osteoarthritis, osteoporosis and fibroticdiseases, dermatosis, including psoriasis, atopic dermatitis andultraviolet radiation (UV)-induced skin damage; autoimmune diseasesincluding systemic lupus eythematosus, multiple sclerosis, psoriaticarthritis, alkylosing spondylitis, tissue and organ rejection,Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes,glomerulonephritis, cancer, including Hodgkins disease, cachexia,inflammation associated with infection and certain viral infections,including acquired immune deficiency syndrome (AIDS), adult respiratorydistress syndrome and Ataxia Telangiestasia

DETAILED DESCRIPTION OF THE INVENTION

The compounds of the present invention are selected from Formula (I)herein below:

-   R₁ represents CONH₂;-   R₂ represents NR₄R₅;-   R₃ is selected from the group consisting of H, CN, CF₃, halogen,    aryl, heteroaryl, alkyl, O-alkyl, and S-alkyl; and can be attached    to either C₄, C₅, C₆ or C₇;-   R₄ represents H or alkyl; and-   R₅ is selected from the group consisting of H, CO-alkyl, SO₂-alkyl,    CONH₂, CONH-alkyl, CONH-aryl, CONH-heteroaryl, CSNH₂, CSNH-alkyl,    CSNH-aryl, CSNH-heteroaryl, SO₂NH₂, SO₂NH-alkyl, SO₂NH-aryl, and    SO₂NH-heteroaryl; or a pharmaceutically acceptable salt thereof.

Preferred compounds useful in the present invention include:

-   2-Amino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylic acid    amide; and-   2-Ureido-benzo[b]thiophene-3-carboxylic acid amide.

This invention provides methods for treating a variety of diseasesassociated with NF-κB activation including inflammatory and tissuerepair disorders; particularly rheumatoid arthritis, inflammatory boweldisease, asthma and COPD (chronic obstructive pulmonary disease)osteoarthritis, osteoporosis and fibrotic diseases; dermatosis,including psoriasis, atopic dermatitis and ultraviolet radiation(UV)-induced skin damage; autoimmune diseases including systemic lupuseythematosus, multiple sclerosis, psoriatic arthritis, alkylosingspondylitis, tissue and organ rejection, Alzheimer's disease, stroke,atherosclerosis, restenosis, diabetes, glomerulonephritis, cancer,including Hodgkins disease, cachexia, inflammation associated withinfection and certain viral infections, including aquired immunedeficiency syndrome (AIDS), adult respiratory distress syndrome, andAtaxia Telangiestasia.

DEFINITIONS

The present invention includes all hydrates, solvates, complexes andprodrugs of the compounds of this invention. Prodrugs are any covalentlybonded compounds, which release the active parent, drug according toFormula I in vivo. If a chiral center or another form of an isomericcenter is present in a compound of the present invention, all forms ofsuch isomer or isomers, including enantiomers and diastereomers, areintended to be covered herein. Inventive compounds containing a chiralcenter may be used as a racemic mixture, an enantiomerically enrichedmixture, or the racemic mixture may be separated using well-knowntechniques and an individual enantiomer may be used alone. In cases inwhich compounds have unsaturated carbon-carbon double bonds, both thecis (Z) and trans (E) isomers are within the scope of this invention. Incases wherein compounds may exist in tautomeric forms, such as keto-enoltautomers, each tautomeric form is contemplated as being included withinthis invention whether existing in equilibrium or predominantly in oneform.

The meaning of any substituent at any one occurrence in Formula I or anysubformula thereof is independent of its meaning, or any othersubstituent's meaning, at any other occurrence, unless specifiedotherwise.

As used herein, “alkyl” refers to an optionally substituted hydrocarbongroup joined by single carbon-carbon bonds and having 1-6 carbon atomsjoined together. The alkyl hydrocarbon group may be linear, branched orcyclic, saturated or unsaturated. Substituents on optionally substitutedalkyl are selected from the group consisting of aryl, OH, O-alkyl, CO,halogen, CF₃, and OCF₃.

As used herein, “aryl” refers to an optionally substituted aromaticgroup with at least one ring having a conjugated pi-electron system,containing up to two conjugated or fused ring systems. Aryl includescarbocyclic aryl, and biaryl groups, all of which may be optionallysubstituted. Substituents are selected from the group consisting ofhalogen, C₁₋₄ alkyl, NH₂, OCF₃, CF₃, O-alkyl, S-alkyl, CN, CHO,SO₂-alkyl and NO₂.

As used herein, “heteroaryl” refers to an optionally substitutedaromatic group with at least one ring having a conjugated pi-electronsystem, containing up to two conjugated or fused ring systems and 1-3heteroatoms selected from O, S and N. Heteroaryl includes carbocyclicheteroarylaryl, aryl-heteroaryl and biheteroarylaryl groups, all ofwhich may be optionally substituted. Preferred aryl include phenyl andnaphthyl. More preferred aryl include phenyl. Preferred substituents areselected from the group consisting of halogen, C₁₋₄ alkyl, NH₂, OCF₃,CF₃, O-alkyl, S-alkyl, CN, CHO, SO₂-alkyl and NO₂. Examples ofheteroaryl rings included pyrrole, furan, thiophene, indole, isoindole,benzofuran, isobenzofuran, benzothiphene, pyridine, quinoline,isoquinoline, quinolizine, pyrazole, imidazole, isoxazole, oxazole,isothiazole, thiazole, pyridazine, pyrimidine, and pyrazine.

As used herein “halogen” refers to include F, Cl, Br, and I.

METHODS OF PREPARATION

The following methods and examples are intended to be illustrative ofthe present invention but not limiting in any way.

The general preparation of analogs of 2-aminobenzothiophene-3-carboxylicacid amide is shown in Scheme I.

The synthesis starts with commercially available ethyl2-amino-4,5,6,7-tetrahydrobenzothiophene-3-carboxylate (1). Protectionof the amino group with acetyl chloride (AcCI) and oxidation with2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) provide benzothiophene3. Deprotection of the acetamide followed by bromination withN-bromosuccinimide (NBS) furnish 2-aminobenzothiophene 5. Palladium(0)mediated Suzuki cross-coupling with boronic acid/ester then afford 6.Reprotection of the amino group with di-tert-butyl dicarbonate [(Boc)₂O]and hydrolysis of the ester group produce acid 8. The resultant acid isactivated with 1,1′-carbonyldiimidazole (CDI), followed by reaction withammonium hydroxide and treatment with trifluoroacetic acid (TFA) thenfurnish 2-amino-benzothiophene-3-carboxylic amide 10. Compound 10 can bereadily transformed to primary urea 11 by reaction with chlorosulfonylisocyanate or to substituted urea 12 by isocyantes (Scheme II).

Urea 15 is synthesized from 2-amino-benzo[b]thiophene-3-carbonitrile(13, Scheme III) employing a two-step procedure; nitrile hydrolysisfollowed by urea formation.

The following examples are intended to be illustrative of the presentinvention but not limiting in anyway.

EXAMPLES AND EXPERIMENTAL

General

Nuclear magnetic resonance spectra were recorded at either 250, 300 or400 MHz using, respectively, a Bruker AM 250, Bruker ARX 300 or BrukerAC 400 spectrometer. CDCl₃ is deuteriochloroform, DMSO-d₆ ishexadeuteriodimethylsulfoxide, and CD₃OD is tetradeuteriomethanol.Chemical shifts are reported in parts per million (d) downfield from theinternal standard tetramethylsilane. Abbreviations for NMR data are asfollows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,dd=doublet of doublets, dt=doublet of triplets, app=apparent, br=broad.J indicates the NMR coupling constant measured in Hertz. Continuous waveinfrared (IR) spectra were recorded on a Perkin-Elmer 683 infraredspectrometer, and Fourier transform infrared (FTIR) spectra wererecorded on a Nicolet Impact 400 D infrared spectrometer. IR and FTIRspectra were recorded in transmission mode, and band positions arereported in inverse wavenumbers (cm⁻¹). Mass spectra were taken oneither VG 70 FE, PE Syx API III, or VG ZAB HF instruments, using fastatom bombardment (FAB) or electrospray (ES) ionization techniques.Elemental analyses were obtained using a Perkin-Elmer 240C elementalanalyzer. Melting points were taken on a Thomas-Hoover melting pointapparatus and are uncorrected. All temperatures are reported in degreesCelsius.

Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layerplates were used for thin layer chromatography. Both flash and gravitychromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh)silica gel.

Where indicated, certain of the materials were purchased from theAldrich Chemical Co., Milwaukee, Wis., TCI America, Portland, Oreg.

Example 1 Preparation of2-amino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylic Acid Amide

1a) 2-Acetylamino-4, 5, 6, 7-tetrahydrobenzothiophene-3-carboxylic AcidEthyl Ester

To a solution of ethyl 2-amino-4, 5, 6,7-tetrahydrobenzothiophene-3-carboxylate (16 g, 71.1 mmol) in THF (100mL) was added 4-(dimethylamino)pyridine (434 mg, 3.55 mmol) and acetylchloride (6.1 mL, 85.3 mmol). After stirring for 2 h at roomtemperature, the solution was diluted with brine solution (500 mL) andextracted with ethyl acetate (500 mL, 3×). The combined organic phaseswere dried over MgSO₄, filtered, and concentrated to provide the titlecompound (18.3 g, 96%) as a light yellow solid: MS (ES) m/z 268 (M+H)⁺.

1b) 2-Acetylamino-benzo[b]thiophene-3-carboxylic Acid Ethyl Ester

A solution of 1a (3.0 g, 11.24 mmol) in benzene (100 mL) was mixed with2,3-dichloro-5,6-dicyano-1,4-benzoquinone (3.8 g, 16.85 mmol). Theresultant mixture was heated at reflux for 2 h, diluted with saturatedNaHCO₃ solution (300 mL) and extracted with ethyl acetate (300 mL, 3×).The combined organic phases were dried over MgSO₄, filtered, andconcentrated. Flash chromatography (hexanes/ethyl acetate, 8:1) thenprovided the title compound (1.06 g, 35%) as a yellow solid: MS (ES) m/z264 (M+H)⁺;

1c) 2-Amino-benzo[b]thiophene-3-carboxylic Acid Ethyl Ester

To a solution of 1b (1.06 g, 4.03 mmol) in toluene (100 mL) was addedpyrrolidine (5 mL). The resultant mixture was heated at 100° C. for 8 h,diluted with brine solution (200 mL) and extracted with ethyl ether (300mL, 3×). The combined organic phases were dried over MgSO₄, filtered,and concentrated. Flash chromatography (hexanes/ethyl acetate, 4:1) thenafforded the title compound (0.76 g, 85%) as a yellow solid: MS (ES) m/z222 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ8.14 (d, J=8.0 Hz, 1H), 7.53 (d,J=7.2 Hz, 1H), 7.28 (m, 1H), 7.04 (m, 1H), 6.51 (brs, 2H), 4.45 (q,J=7.2 Hz, 2H), 1.50 (t, J=7.2 Hz, 3H).

1d) 2-Amino-6-bromo-benzo[b]thiophene-3-carboxylic Acid Ethyl Ester

To a solution of 1c (760 mg, 3.44 mmol) in CHCl₃ (10 mL) was addedN-bromosuccinimide (673 mg, 3.78 mmol). The resultant mixture wasstirred for 1 h, then mixed with saturated NaHCO₃ solution (100 mL), andextracted with methylene chloride (100 mL, 3×). The combined organicphases were dried over MgSO₄, filtered, and concentrated. Flashchromatography (hexanes/ethyl acetate, 4:1) then provided the titlecompound (930 mg, 90%) as a yellow solid: MS (ES) m/z 300 (M+H)⁺; ¹H NMR(400 MHz, CDCl₃) δ7.96 (d, J=8.8 Hz, 1H), 7.63 (s, 1H), 7.42 (d, J=8.8Hz, 1H), 6.52 (brs, 2H), 4.42 (q, J=7.2 Hz, 2H), 1.48 (t, J=7.2 Hz, 3H).

1e) 2-Amino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylic AcidEthyl Ester

To a solution of 1d (200 mg, 0.67 mmol) in 1,4-dioxane/water (40 mL,3:1) was added 4-fluorophenylboronic acid (209 mg, 1.34 mmol), NaHCO₃(225 mg, 2.68 mmol) and Pd(PPh₃)₄ (9% Pd, 79 mg, 0.067 mmol). Theresultant mixture was heated at 110° C. for 1 h, diluted with brinesolution (50 mL) and extracted with ethyl acetate (50 mL, 3×). Thecombined organic phases were dried over MgSO₄, filtered, andconcentrated. Flash chromatography (hexanes/ethyl acetate, 4:1) thenprovided the title compound (190 mg, 90%) as a pink sticky oil: MS (ES)m/z 316 (M+H)⁺; ¹H NMR (400 MHz, CDCl₃) δ8.16 (d, J=8.4 Hz, 1H), 7.69(s, 1H), 7.62 (m, 2H), 7.50 (d, J=8.4 Hz, 1H), 7.15 (m, 2H), 6.58 (brs,2H), 4.46 (q, J=7.2 Hz, 2H), 1.50 (t, J=7.2 Hz, 3H).

1f)2-tert-Butoxycarbonylamino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylicAcid Ethyl Ester

To a solution of 1e (190 mg, 0.603 mmol) in THF (10 mL) was added4-(dimethylamino)pyridine (7.4 mg, 0.06 mmol) and di-tert-butyldicarbonate (158 mg, 0.724 mmol). The resultant mixture was stirred atroom temperature for 1 h, diluted with brine solution (50 mL), andextracted with ethyl acetate (50 mL, 3×). The combined organic phaseswere dried over MgSO₄, filtered, and concentrated. Flash chromatography(hexanes/ethyl acetate, 4:1) then provided the title compound (200 mg,80%) as a yellow solid: MS (ES) m/z 416 (M+H)⁺.

1g)2-tert-Butoxycarbonylamino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylicAcid

A solution of 1f (80 mg, 0.193 mmol) in ethanol/water (1:1, 10 mL) wasmixed with KOH (21.6 mg, 0.39 mmol). The resultant mixture was heated at60° C. for 1 h, diluted with HCl (30 mL, 1N), and extracted with ethylacetate (30 mL, 3×). The combined organic phases were dried over MgSO₄,filtered, and concentrated to afford the title compound (62 mg, 84%) asa white solid: MS (ES) m/z 388 (M+H)⁺.

1h) [3-Carbamoyl-6-(4-fluoro-phenyl)-benzo[b]thiophene-2-yl]-carbamicAcid Tert-butyl Ester

To a solution of 1g (150 mg, 0.38 mmol) in DMF (3 mL) was added1,1′-carbonyl diimidazole (125 mg, 0.78 mmol). The resultant solutionwas stirred at room temperature for 1 h and mixed with ammoniumhydroxide (37%, 5 mL). The mixture was diluted with brine solution (10mL) and extracted with ethyl acetate (20 mL, 3×). The combined organicphases were dried over MgSO₄, filtered, and concentrated. Flashchromatography (hexanes/ethyl acetate, 1:1) then provided the titlecompound (48 mg, 32%) as a white solid: MS (ES) m/z 387 (M+H)⁺; ¹H NMR(400 MHz, CDCl₃) δ11.14 (s, 1H), 7.94 (s, 1H), 7.85 (d, J=4.4 Hz, 1H),7.61 (m, 3H), 7.18 (m, 2H), 5.87 (brs, 2H), 1.59 (s, 9H).

1i) 2-Amino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylic AcidAmide

A solution of 1h (20 mg, 0.051 mmol) in CH₂Cl₂ (5 mL) was mixed withtrifluoroacetic acid (0.5 mL). The resultant solution was stirred atroom temperature for 1 h, then mixed with saturated NaHCO₃ solution (30mL), and extracted with ethyl acetate (30 mL, 3×). The combined organicphases were dried over MgSO₄, filtered, and concentrated. Flashchromatography (hexanes/ethyl acetate, 1:1) provided the title compound(8 mg, 55%) as a white solid: MS (ES) m/z 287 (M+H)⁺; ¹H NMR (400 MHz,CD₃OD) δ7.73 (s, 1H), 7.63-7.50 (m, 3H), 7.42 (m, 1H), 7.08 (m, 2H).

Example 2 Preparation of 2-ureido-benzo[b]thiophene-3-carboxylic AcidAmide

2a) 2-Amino-benzo[b]thiophene-3-carboxylic Acid Amide

A solution of 2-amino-benzo[b]thiophene-3-carbonitrile (50 mg, 0.29mmol) in concentrated H₂SO₄ (1.5 mL) was heated at 60° C. for 2 h. Thesolution was poured into ice-water (5 mL), mixed with saturated NaHCO₃solution (30 mL), and extracted with ethyl acetate (30 mL, 3×). Thecombined organic phases were dried over MgSO₄, filtered, andconcentrated. Flash chromatography (hexanes/ethyl acetate, 1:1) thenprovided the title compound (23 mg, 41%) as a white solid: MS (ES) m/z193 (M+H)⁺.

2b) 2-Ureido-benzo[b]thiophene-3-carboxylic Acid Amide

To the mixture of 2a (20 mg, 0.104 mmol) in CH₂Cl₂ (10 mL) was addedchlorosulfonyl isocyanate (15 μL, 0.15 mmol). The resultant mixture wasstirred at room temperature for 1 h and mixed with water (0.5 mL).Separation via a reverse phase HPLC provided the title compound (10 mg,40%) as a white solid: MS (ES) m/z 236 (M+H)⁺; ¹H NMR (400 MHz, DMSO-d₆)δ10.77 (s, 1H), 7.84 (m, 2H), 7.56 (brs, 3H), 7.20 (m, 1H), 7.08 (m,1H), 6.98 (brs, 1H).

This invention provides a pharmaceutical composition, which comprises acompound according to Formula, I and a pharmaceutically acceptablecarrier, diluent or excipient. Accordingly, the compounds of Formula Imay be used in the manufacture of a medicament. Pharmaceuticalcompositions of the compounds of Formula I prepared as hereinbeforedescribed may be formulated as solutions or lyophilized powders forparenteral administration. Powders may be reconstituted by addition of asuitable diluent or other pharmaceutically acceptable carrier prior touse. The liquid formulation may be a buffered, isotonic, aqueoussolution. Examples of suitable diluents are normal isotonic salinesolution, standard 5% dextrose in water or buffered sodium or ammoniumacetate solution. Such formulation is especially suitable for parenteraladministration, but may also be used for oral administration orcontained in a metered dose inhaler or nebulizer for insufflation. Itmay be desirable to add excipients such as polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol,sodium chloride or sodium citrate.

Alternately, these compounds may be encapsulated, tableted or preparedin an emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule.

Typical compositions for inhalation are in the form of a dry powder,solution, suspension or emulsion. Administration may for example be bydry powder inhaler (such as unit dose or multi-dose inhaler, e.g. asdescribed in U.S. Pat. No. 5,590,645 or by nebulisation or in the formof a pressurized aerosol. Dry powder compositions typically employ acarrier such as lactose, trehalose or starch. Compositions fornebulisation typically employ water as vehicle. Pressurized aerosolstypically employ a propellant such as dichlorodifluoromethane,trichlorofluoromethane or, more preferably, 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoro-n-propane or mixtures thereof. Pressurizedaerosol formulations may be in the form of a solution (perhaps employinga solubilising agent such as ethanol) or a suspension which may beexcipient free or employ excipients including surfactants and/orco-solvents (e.g. ethanol). In dry powder compositions and suspensionaerosol compositions the active ingredient will preferably be of a sizesuitable for inhalation (typically having mass median diameter (MMD)less than 20 microns e.g. 1-10 especially 1-5 microns). Size reductionof the active ingredient may be necessary e.g. by micronisation.

Pressurized aerosol compositions will generally be filled into canistersfitted with a valve, especially a metering valve. Canisters mayoptionally be coated with a plastics material e.g. a fluorocarbonpolymer as described in WO96/32150. Canisters will be fitted into anactuator adapted for buccal delivery.

Typical compositions for nasal delivery include those mentioned abovefor inhalation and further include non-pressurized compositions in theform of a solution or suspension in an inert vehicle such as wateroptionally in combination with conventional excipients such as buffers,anti-microbials, tonicity modifying agents and viscosity modifyingagents which may be administered by nasal pump.

For rectal administration, the compounds of this invention may also becombined with excipients such as cocoa butter, glycerin, gelatin orpolyethylene glycols and molded into a suppository.

The methods of the present invention include topical inhaled andintracolonic administration of the compounds of Formula I. By topicaladministration is meant non-systemic administration, including theapplication of a compound of the invention externally to the epidermis,to the buccal cavity and instillation of such a compound into the ear,eye and nose, wherein the compound does not significantly enter theblood stream. By systemic administration is meant oral, intravenous,intraperitoneal and intramuscular administration. The amount of acompound of the invention (hereinafter referred to as the activeingredient) required for therapeutic or prophylactic effect upon topicaladministration will, of course, vary with the compound chosen, thenature and severity of the condition being treated and the animalundergoing treatment, and is ultimately at the discretion of thephysician

While it is possible for an active ingredient to be administered aloneas the raw chemical, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.01 to 5.0 wt %. of the formulation.

The topical formulations of the present invention, both for veterinaryand for human medical use, comprise an active ingredient together withone or more acceptable carriers therefor and optionally any othertherapeutic ingredients. The carrier must be “acceptable” in the senseof being compatible with the other ingredients of the formulation andnot deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required such as: liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable container,which is then sealed and sterilized by autoclaving, or maintaining at90-100 C for half an hour. Alternatively, the solution may be sterilizedby filtration and transferred to the container by an aseptic technique.Examples of bactericidal and fungicidal agents suitable for inclusion inthe drops are phenylmercuric nitrate or acetate (0.002%), benzalkoniumchloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solventsfor the preparation of an oily solution include glycerol, dilutedalcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient in finelydivided or powdered form, alone or in solution or suspension in anaqueous or non-aqueous fluid, with the aid of suitable machinery, with agreasy or non-greasy basis. The basis may comprise hydrocarbons such ashard, soft or liquid paraffin, glycerol, beeswax, a metallic soap, amucilage, an oil of natural origin such as almond, corn, arachis, castoror olive oil, wool fat or its derivatives, or a fatty acid such asstearic or oleic acid together with an alcohol such as propylene glycolor macrogols. The formulation may incorporate any suitable surfaceactive agent such as an anionic, cationic or non-ionic surface activeagent such as sorbitan esters or polyoxyethylene derivatives thereof.Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredientssuch as lanolin, may also be included.

UTILITY OF THE PRESENT INVENTION

The compounds of Formula I are useful as inhibitors of the IKK-betakinase phosphorylation of IκB and as such are inhibitors of NF-κBactivation. The present method utilizes compositions and formulations ofsaid compounds, including pharmaceutical compositions and formulationsof said compounds.

The present invention particularly provides methods of treatment ofdiseases associated with inappropriate NF-κB activation, which methodscomprise administering to an animal, particularly a mammal, mostparticularly a human in need thereof one or more compounds of Formula I.The present invention particularly provides methods for treatinginflammatory and tissue repair disorders, particularly rheumatoidarthritis, inflammatory bowel disease, asthma and COPD (chronicobstructive pulmonary disease) osteoarthritis, osteoporosis and fibroticdiseases; dermatosis, including psoriasis, atopic dermatitis andultraviolet radiation (UV)-induced skin damage, autoimmune diseasesincluding systemic lupus eythematosus, multiple sclerosis, psoriaticarthritis, alkylosing spondylitis, tissue and organ rejection,Alzheimer's disease, stroke, atherosclerosis, restenosis, diabetes,glomerulonephritis, cancer, including Hodgkins disease, cachexia,inflammation associated with infection and certain viral infections,including aquired immune deficiency syndrome (AIDS), adult respiratorydistress syndrome and Ataxia Telangiestasia.

For acute therapy, parenteral administration of one or more compounds ofFormula I is useful. An intravenous infusion of the compound in 5%dextrose in water or normal saline, or a similar formulation withsuitable excipients, is most effective, although an intramuscular bolusinjection is also useful. Typically, the parenteral dose will be about0.01 to about 50 mg/kg; preferably between 0.1 and 20 mg/kg, in a mannerto maintain the concentration of drug in the plasma at a concentrationeffective to inhibit IKK-beta and therefore activation of NF-κB. Thecompounds are administered one to four times daily at a level to achievea total daily dose of about 0.4 to about 80 mg/kg/day. The preciseamount of a compound used in the present method which is therapeuticallyeffective, and the route by which such compound is best administered, isreadily determined by one of ordinary skill in the art by comparing theblood level of the agent to the concentration required to have atherapeutic effect.

The compounds of Formula I may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto inhibit IKK-beta and therefore activation of NF-κB or to achieve anyother therapeutic indication as disclosed herein. Typically, apharmaceutical composition containing the compound is administered at anoral dose of between about 0.1 to about 50 mg/kg in a manner consistentwith the condition of the patient. Preferably the oral dose would beabout 0.5 to about 20 mg/kg.

The compounds of Formula I may also be administered topically to thepatient, in a manner such that the concentration of drug is sufficientto inhibit IKK-beta and therefore activation of NF-κB or to achieve anyother therapeutic indication as disclosed herein. Typically, apharmaceutical composition containing the compound is administered in atopical formulation of between about 0.01% to about 5% w/w.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

The ability of the compounds described herein to inhibit the activationof NF-κB is clearly evidenced in their ability to inhibit thephosphorylation of the N-terminal fragment of IκB-α (by IKK-β(see Table1 for examples). These compounds also block the degradation of IκB-α andthe nuclear translocation of NF-κB in human monocyctes and othermammalian cells upon activation of the cells with a pro-inflammatorystimulii (e.g., TNF-α, LPS, etc.). In addition these compounds inhibitpro-inflammatory mediator production from LPS-stimulated human monocytesand stimulated human primary synovial fibroblasts. The utility of thepresent NF-κB inhibitors in the therapy of diseases is premised on theimportance of NF-κB activation in a variety of diseases.

NF-κB plays a key role in the regulated expression of a large number ofpro-inflammatory mediators including cytokines such as TNF, IL-1β, IL-6and IL-8 (Mukaida et al., 1990; Liberman and Baltimore, 1990; Matsusakaet al., 1993), cell adhesion molecules, such as ICAM and VCAM (Marui etal., 1993; Kawai et al., 1995; Ledebur and Parks, 1995), and induciblenitric oxide synthase (iNOS) (Xie et al., 1994; Adcock et al., 1994).(Full reference citations are at the end of this section). Suchmediators are known to play a role in the recruitment of leukocytes atsites of inflammation and in the case of iNOS, may lead to organdestruction in some inflammatory and autoimmune diseases(McCartney-Francis et al., 1993; Kleemann et al., 1993.

Evidence for an important role of NF-κB in inflammatory disorders isobtained in studies of asthmatic patients. Bronchial biopsies taken frommild atopic asthmatics show significant increases in the number of cellsin the submucosa staining for activated NF-κB, total NF-κB, andNF-κB-regulated cytokines such as GM-CSF and TNFα compared to biopsiesfrom normal non-atopic controls (Wilson et al., 1998). Furthermore, thepercentage of vessels expressing NF-κB immunoreactivity is increased asis IL-8 immunoreactivity in the epithelium of the biopsy specimens(Wilson et al., 1998). As such, inhibition of IL-8 production throughthe inhibition of NF-κB, as has been demonstrated by these compoundswould be predicted be beneficial in airway inflammation.

Recent studies suggest that NF-κB may also play a critical role in thepathogenesis of inflammatory bowel disease (IBD). Activated NF-κB isseen in colonic biopsy specimens from Chron's disease and ulcerativecolitis patients (Ardite et al., 1998; Rogler et al., 1998; Schreiber etal., 1998). Activation is evident in the inflamed mucosa but not inuninflamed mucosa (Ardite et al., 1998; Rogler et al., 1998) and isassociated with increased IL-8 mRNA expression in the same sites (Arditeet al., 1998). Furthermore, corticosteroid treatment strongly inhibitsintestinal NF-κB activation and reduces colonic inflammation (Ardite etal., 1998; Schreiber et al., 1998). Again, inhibition of IL-8 productionthrough the inhibition of NF-κB, as has been demonstrated by thesecompounds would be predicted be beneficial in inflammatory boweldisease.

Animal models of gastrointestinal inflammation provide further supportfor NF-κB as a key regulator of colonic inflammation. Increased NF-κBactivity is observed in the lamina propria macrophages in2,4,6,-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice withp65 being a major component of the activated complexes (Neurath et al.,1996; Neurath and Pettersson, 1997). Local administration of p65antisense abrogates the signs of established colitis in the treatedanimals with no signs of toxicity (Neurath et al., 1996; Neurath andPettersson, 1997). As such, one would predict that small moleculeinhibitors of NF-κB would be useful in the treatment of IBD.

Further evidence for a role of NF-κB in inflammatory disorders comesfrom studies of rheumatoid synovium. Although NF-κB is normally presentas an inactive cytoplasmic complex, recent immunohistochemical studieshave indicated that NF-κB is present in the nuclei, and hence active, inthe cells comprising human rheumatoid synovium (Handel et al., 1995;Marok et al., 1996; Sioud et al., 1998) and in animal models of thedisease (Tsao et al., 1997). The staining is associated with type Asynoviocytes and vascular endothelium (Marok et al., 1996). Furthermore,constitutive activation of NF-κB is seen in cultured synoviocytes(Roshak et al., 1996; Miyazawa et al., 1998) and in synovial cellcultures stimulated with IL-1 or TNFα (Roshak et al., 1996; Fujisawa etal., 1996; Roshak et al., 1997). Thus, the activation of NF-κB mayunderlie the increased cytokine production and leukocyte infiltrationcharacteristic of inflamed synovium. The ability of these compounds toinhibit NF-κB and thereby inhibit the production of pro-inflammatorymediators (e.g. cytokines and prostanoids) by these cells would bepredicted to yield benefit in rheumatoid arthritis.

Biological Assays

The compounds of this invention may be tested in one of severalbiological assays to determine the concentration of compound, which isrequired to have a given pharmacological effect.

NF-κB activity may also be measured in an electrophoretic mobility shiftassay (EMSA) to assess the presence of NF-κB protein in the nucleus. Thecells of interest are cultured to a density of 1×10⁶/mL. The cells areharvested by centrifugation, washed in PBS without Ca²⁺ and Mg²⁺ andresuspended in PBS with Ca²⁺ and Mg²⁺ at 1×10⁷ cells/mL. To examine theeffect of compound on the activation of NF-κB, the cell suspensions aretreated with various concentrations of drug or vehicle (DMSO, 0.1%) for30 min. at 37° C. prior to stimulation with TNF-α (5.0 ng/mL) for anadditional 15 min. Cellular and nuclear extracts are prepared follows.Briefly, at the end of the incubation period the cells (1×10⁷ cells) arewashed 2× in PBS without Ca²⁺ and Mg²⁺. The resulting cell pellets areresuspended in 20 uL of Buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 1.5mM MgCl₂, 0.5 mM dithiothreitol (DTT) and 0.1% NP-40) and incubated onice for 10 min. The nuclei are pelleted by microcentrifugation at 3500rpm for 10 min at 4° C. The resulting supernatant was collected as thecellular extract and the nuclear pellet was resuspended in 15 uL BufferC (20 mM Hepes (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl₂, 25% glycerol, 0.2 mMEDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF)). Thesuspensions are mixed gently for 20 min at 4° C. then microcentrifugedat 14,000 rpm for 10 min at 4° C. The supernatant is collected anddiluted to 60 uL with Buffer D (20 mM Hepes (pH 7.9), 50 mM KCl, 20%glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). All samples arestored at −80° C. until analyzed. The protein concentration of theextracts is determined according to the method of Bradford (Bradford,1976) with BioRad reagents.

The effect of compounds on transcription factor activation is assessedin an electrophoretic mobility shift assay (EMSA) using nuclear extractsfrom treated cells as described above. The double stranded NF-κBconsensus oligonucleotides (5′-AGTTGAGGGGACTTTCCCAGGC-3′) are labelledwith T₄ polynucleotide kinase and [g-³²P]ATP. The binding mixture (25uL) contains 10 mM Hepes-NaOH (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mMKCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.3 mg/mL bovineserum albumin, and 1 ug poly(dI-dC)∘poly(dI-dC). The binding mixtures(10 ug nuclear extract protein) are incubated for 20 min at roomtemperature with 0.5 ng of ³²P-labelled oligonucleotide (50,000-100,000cpm) in the presence or absence of unlabeled competitor after which themixture is loaded on a 4% polyacrylamide gel prepared in 1× Trisborate/EDTA and electrophoresed at 200 V for 2 h. Followingelectrophoresis the gels are dried and exposed to film for detection ofthe binding reaction.

The effect of compounds on the phosphorylation of IκB may be monitoredin a Western blot. Cellular extracts are subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels(BioRad, Hercules, Calif.) and the proteins transferred tonitrocellulose sheets (Hybond™-ECL, Amersham Corp., Arlington Heights,Ill.). Immunoblot assays are performed using a polyclonal rabbitantibody directed against IκBα or IκBβ followed with aperoxidase-conjugated donkey anti-rabbit secondary antibody (AmershamCorp., Arlington Heights, Ill.). Immunoreactive bands are detected usingthe Enchanced Chemiluminescence (ECL) assay system (Amersham Corp.,Arlington Heights, Ill.).

Assays for IκB kinases were conducted as follows: IKK-α was expressed asa hexa-histidine tagged protein in baculovirus-infected insect cells andpurified over a Ni-NTA affinity column. Kinase activity was assayedusing 50 ng of purified protein in assay buffer (20 mM Hepes, pH 7.7, 2mM MgCl₂, 1 mM MnCl₂, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP,0.3 mM Na₃VO₄, 1 mM benzamidine, 2 μM PMSF, 10 μg/ml aprotinin, 1 ug/mLleupeptin, 1 ug/mL pepstatin, 1 mM DTT) containing variousconcentrations of compound or DMSO vehicle and ATP as indicated(Pharmacia Biotech Inc., Piscataway, N.J.). The reaction was started bythe addition of 200 ng IκB-GST (Santa Cruz Biotechnology, Inc., SantaCruz, Calif.), in a total volume of 50 uL. The reaction was allowed toproceed for 1 h. at 30° C. after which the reaction was terminated bythe addition of EDTA to a final concentration of 20 mM. Kinase activitywas determined by dissociation-enhanced lanthanide fluorescenceimmunoassay (Wallac Oy, Turku, Finland) using a phospho-IκB-α (Ser32)antibody (New England Biolabs, Inc., Beverly, Mass.) and anEu³⁺-labelled anti-rabbit IgG (Wallac Oy, Turku, Finland). The plateswere read in a VICTOR 1420 Multilabel Counter (Wallac), using a standardeuropium protocol (excitation 340 nm, emission 615 nm; fluorescencemeasured for 400 μs after a 400 usec delay). Data are expressed asfluorescence (cps) units.

IKK-β was expressed as a GST-tagged protein, and its activity wasassessed in a 96-well scintillation proximity assay (SPA). Briefly,IKK-β was diluted in assay buffer as described above (20 nM final), withvarious concentrations of compound or DMSO vehicle, 240 nM ATP and 200nCi [γ-³³P]-ATP (10 mCi/mL, 2000 Ci/mmol; NEN Life Science Products,Boston, Mass.). The reaction was started with the addition of abiotinylated peptide comprising amino acids 15-46 of IκB-α (AmericanPeptide) to a final concentration of 2.4 μM, in a total volume of 50 uL.The sample incubated for one hour a 30° C., followed by the addition of150 uL of stop buffer (PBS w/o Ca²⁺, Mg²⁺, 0.1% Triton X-100 (v/v), 10mM EDTA) containing 0.2 mg streptavidin-coated SPA PVT beads (AmershamPharmacia Biotech, Piscataway, N.J.). The sample was mixed, incubatedfor 10 min. at room temperature, centrifuged (1000×g, 2 minutes), andmeasured on a Hewlett-Packard TopCount.

In addition, IKK-β or IKK-α activity is measured by phosphorylation ofrecombinant GST-IkappaBalpha using time-resolved fluorescence resonanceenergy transfer (TR-FRET) in 384-well microtitre plates. Briefly IKK-βorIKK-α is diluted in assay buffer (50 mM HEPES pH 7.4 containing 10 mMmagnesium chloride, 1 mM CHAPS, 1 mM DTT and 0.01% w/v BSA) to 5 nMfinal concentration. This is added to various concentrations of compoundor DMSO vehicle and the reaction started by addition of 25 nMGST-IkappaBalpha and 1 μM ATP in assay buffer to a volume of 30 uL.After incubation for 30 min at ambient temperature the reaction wasstopped by addition of 50 mM pH 7.4 EDTA (15 uL). Detection ofphophorylated product was achieved by addition of a LANCE europiumchelate labelled specific anti-phosphoserine monoclonal antibody at 0.5nM final concentration (Cell signalling Technology via Perkin Elmer) andallophycocyanin labelled anti-GST antibody at 10 nM final concentration(Prozyme) to give a final volume of 60 μl. After a further incubation atambient temperature of a least 30 min the signal was read on a PerkinElmer Discovery fluorimeter.

The effect of IKK-β inhibitors on primary synovial fibroblast mediatorproduction was assesses as follows: Primary cultures of human RSF wereobtained by enzymatic digestion of synovium obtained from adult patientswith rheumatoid arthritis as previously described (Roshak et al.,1996b). Cells were cultured in Earl's Minimal Essential Medium (EMEM)which contained 10% fetal bovine serum (FBS), 100 units/ml penicillinand 100 μg/ml streptomycin (GIBCO, Grand Island, N.Y.), at 37° C. and 5%CO₂. Cultures were used at passages 4 through 9 in order to obtain amore uniform type B fibroblast population. For some studies, fibroblastswere plated at 5×10⁴ cells/mL in 16 mm (diameter) 24 well plates(Costar, Cambridge, Mass.). Cells (70-80% confluence) were exposed toIL-1β (1 ng/mL) (Genzyme, Cambridge, Mass.) for the designated time.Drugs in DMSO vehicle (1%) were added to the cell cultures 15 minutesprior to the addition of IL-1. Studies were conducted 3-4 times usingsynovial cells from different donors. RSF cellular extracts wereprepared from cells treated as described above. Briefly, human RSF wereremoved by trypsin/EDTA, washed, and harvested by centrifugation.Cellular extracts were prepared as previously described (Dignarn et al.,1983; Osborn, et al., 1989). Briefly, at the end of the incubationperiod the cells (1×10⁷ cells) were washed 2× in PBS without Ca²⁺ andMg²+. The resulting cell pellets were resuspended in 20 uL of Buffer A(10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM.

Effect of IKK-β inhibition on human monocyte stimulated eicosanoid andcytokine production was assessed as follows: Monocytes were isolatedfrom heparinized whole blood by double gradient centrifugation aspreviously described. Isolated monocyte enriched PBMCs were then adheredto 24 well culture plates at 2×10⁶ cells/mL in RPMI 1640 10% FBS(Hyclone, Logan, Utah) for 2 h. to further enrich the monocytepopulation. The media was then removed, cells washed once with RPMI1640, and 1 mL RPMI 1640 10% FBS was added to the wells. Test compoundsare added to the wells with a final vehicle concentration of 0.05% DMSO.Monocytes were activated by the addition of 200 ng/mL endotoxin (LPS; E.coli serotype 026:B6)(Sigma, St. Louis, Mo.) and incubated for 24 hrs.Cell-free supernates were analyzed by ELISA for TNF-α (EIA developed atSB), PGE₂ (Cayman Chemical, Ann Arbor, Mich.), and IL-8 and IL-6Biosource International, Camarillo, Calif.). Viability of the cells wasdetermined by trypan blue exclusion.

Effect of IKK-β inhibitors on phorbol ester-induced inflammation wasassessed as follows: The inflammatory response induced by the cutaneousapplication of phorbol ester (PMA) to the external pinnae of Balb/c micehas proven to be a useful model to examine multifactorial inflammatorycell infiltration and inflammatory alteration of epidermis. The intenseinflammatory lesion is dominated by neutrophil infiltration, which canbe easily quantified by measurement tissue concentrationmyeloperoxidase, an azuriphilic granular enzyme present in neutrophils.In addition, the overall intensity of the inflammatory response can bemeasured by determination of ear thickness. Balb/c mice (n=6/group) wereadministered drug treatment or vehicle followed by PMA (4 ug/ear). Themice were sacrificed 4 h. later, the ear thickness determined and NF-κBactivation was monitored by IκBα western or EMSA analysis.

Effect of IKK-β inhibitors on rat carrageenan-induced paw edema wasassessed as follows: Male Lewis rats (Charles River, Raleigh, N.C.) werehoused and allowed free access to food and water, and weighed between200-275 g for each experiment. Compound or vehicle (0.5% Tragacanth(p.o.) or 10% DMSO, 5% DMA, 30% Cremophor(i.p.)) was administered 30minutes to 1 hour prior to the carrageenan injection. Edema was inducedby injection of 1% carrageenan in sterile dH2O (0.05 ml/paw) into theplantar surface of the right hindpaw. Paw thickness was measured priorto administration of compound or vehicle, and again at 3 hours, todetermine change in paw volume. Rats were euthanized by CO2 inhalationand the right hindfoot was removed, immediately frozen in liquidnitrogen and stored at −80 C for analysis.

To determine the effects of an IKK-2 inhibitor in the mousecollagen-induced arthritis (CIA) model, 12 male DBA/1 mice (20-22 grams)per treatment group were immunized on day 0 with a total of 100 uL ofcomplete Freund's adjuvant (CFA) containing 200 ug of bovine type IIcollagen. On day 21 mice were boosted with 100 uL of phosphate bufferedsaline (PBS) containing 200 ug of bovine type II collagen (the 100 uL ofcollagen/CFA or collagen/PBS was injected subcutaneously into the tail).The IKK-2 inhibitor in vehicle, or vehicle alone, was administeredintraperitoneally, twice daily, from days 1 through 40 (disease symptomsare evident beginning on days 25-28). Two additional treatment groupsincluded the positive control etanercept (Enbrel) (4 mg/kg,intraperitoneally, every other day), and the etanercept vehicle (PBS).Mice were scored daily, through day 50, for clinical symptoms (seebelow), and paw thicknesses were measured. In addition to the 12 miceper treatment group that were scored throughout the experiment, atseveral time points during the course of disease satellite mice (3-5 pertreatment group) treated as described above were utilized to measurecytokine/chemokine levels and p65 levels in the paw, the ex vivo antigenrecall response by draining lymph node antigen recall response bydraining lymph node

Induction of arthritis AIA is induced by a single injection of 0.75 mgof Mycobacterium butyricum (Difco, Detroit, Mich.) suspended in paraffinoil into the base of the tail of male Lewis rats aged 6-8 weeks (160-180g). Hindpaw volumes are measured by a water displacement method on day16 and/or day 20. Test compounds were homogenized in a suitable vehicleand administered by a suitable route. Control animals are administeredvehicles alone. Two dosing protocols are genrally used: prophylacticdosing, which is initiated on the day of adjuvant injection andtherapeutic administration, initiated on day 10 once inflammation hasbeen established.Clinical ScoringEach paw was assigned a score ranging from 0-4, based on the followingcriteria:

-   0=no inflammation-   1=single swollen digit-   2=several swollen digits, mild paw swelling-   3=several swollen digits, moderate paw swelling-   4=all digits swollen, severe paw swelling

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The above description fully discloses the invention including preferredembodiments thereof. Modifications and improvements of the embodimentsspecifically disclosed herein are within the scope of the followingclaims. Without further elaboration, it is believed that one skilled inthe area can, using the preceding description, utilize the presentinvention to its fullest extent. Therefore the Examples herein are to beconstrued as merely illustrative and not a limitation of the scope ofthe present invention in any way. The embodiments of the invention inwhich an exclusive property or privilege is claimed are defined asfollows.

1. A compound according to formula (I) hereinbelow: (1) herein below:

R₁ represents CONH₂; R₂ represents NR₄R₅; R₃ is selected from the groupconsisting of H, CN, CF₃, halogen, aryl, heteroaryl, alkyl, O-alkyl, andS-alkyl; and can be attached to either C₄, C₅, C₆ or C₇; R₄ represents Hor alkyl; and R₅ is selected from the group consisting of H, CO-alkyl,SO₂-alkyl, CONH₂, CONH-alkyl, CONH-aryl, CONH-heteroaryl, CSNH₂,CSNH-alkyl, CSNH-aryl, CSNH-heteroaryl, SO₂NH₂, SO₂NH-alkyl, SO₂NH-aryl,and SO₂NH-heteroaryl; or a pharmaceutically acceptable salt thereof. 2.A compound according to claim 1 wherein the compound is selected fromthe group consisting of:2-Amino-6-(4-fluoro-phenyl)-benzo[b]thiophene-3-carboxylic acid amide;and 2-Ureido-benzo[b]thiophene-3-carboxylic acid amide.